Oral Biology Archives
Volume 81,
September 2017
, pages 21-25
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https://doi.org/10.1016/j.archoralbio.2017.04.011Get rights and content
Abstract
Saliva is an attractive source for oral microbial detection and quantification, as sampling is non-invasive and rapid.
Goals
Determine whether different saliva preparation methods or preservation time periods affect DNA stability.
Methods
Saliva samples from 4 healthy adult volunteers were processed to obtain 3 different preparations: whole saliva and pellet after centrifugation and supernatant. The purified DNA (MasterPure™) from each sample was divided into 4 aliquots, one for immediate analysis and 3 (stored at -80°C) for later analyzes after 1 week and 2 and 6 months. DNA concentrations and qPCR-based amounts ofPorphyromonas gingivalis,Prevotella intermedia,Parvimonas micra,Fusobacterium nucleatum, Filifactor alocisand Streptococcus mutans were determined.
Results
DNA concentration did not decrease (P>0.05) over the 6 month period in any sample. Mean (SE) concentrations of DNA (ng/μl) in whole saliva were 152.2 (51.2) and 147.8 (50) at day 0 and 6 months, respectively. Likewise, the values for the pellet were 134.9 (42.5) and 133.6 (42.9), and for the supernatant, 11 (1.9) and 8.9 (2.3), with the difference being significant (P<0.001) between supernatant and whole saliva or pellet. The amounts of most bacterial species found on day 0 remained stable over the 6 month period in all saliva preparations. In the supernatant, the amounts of species were lower (P<0.05) than in whole saliva or pellet.
Conclusions
DNA concentrations were comparable between whole and pellet saliva, suggesting that either could be used for DNA-based analyses. Our results also demonstrated that DNA extracted from saliva can be preserved at -80°C for at least 6 months without decreasing DNA concentration.
Introduction
Saliva contains a variety of native bacteria released from biofilms that grow on soft and hard oral surfaces. The whole saliva sampling procedure is non-invasive and quick and does not require specialized skills or equipment. Easy access has led to an extensive research effort to explore the possibilities of using saliva for various diagnostic purposes, including detection and quantification of salivary bacteria (Sakamoto, Takeuchi, Umeda, Ishikawa, and Benno, 2001; von Troil-Linden, Torkko , Alaluusua, Jousimies-Somer, & Asikainen, 1995; Zhou et al., 2015). Both culture and DNA-based methods have revealed the presence of bacteria associated with periodontitis and caries in saliva (Asikainen, Alaluusua, & Saxen, 1991; Darout, Albandar, Skaug, & Ali, 2002; Kononen, Jousimies-Somer, & Asikainen, 1994; Umeda, Contreras, Chen, Bakker, & Slots, 1998). Furthermore, salivary levels of these species have been associated with increased risk of disease severity (Slots and Slots, 2011, Umeda et al., 1998, von Troil-Linden et al., 1995). Little is known about the prognostic value of the presence or quantity of these bacteria in saliva.
DNA-based methods have gained popularity among researchers due to their straightforward laboratory protocols that are simpler, faster, and more discriminative than culture for bacterial identification. Preparation of saliva sample for DNA extraction can affect DNA yield and detection of target molecules. Total saliva or, after centrifugation, the pellet has been used for DNA extraction (Durdiakova, Kamodyova, Ostatnikova, Vlkova, & Celec, 2012; Ng, Koh, Choo, Ng, & Fu, 2004) more frequently than the respective supernatant (Gu et al., 2009; Li, Zhou, St John and Wong, 2004; Vandewoestyne, Van Hoofstat, Franssen, Van Nieuwerburgh and Deforce, 2013). Since free DNA is released from live bacteria (Draghi & Turner, 2006) and lysed in the extracellular medium, saliva supernatant can also serve as material for the analysis of oral bacteria by DNA-based techniques. No DNA yield comparisons of various salivary biolayers were found.
In clinical studies, extracted DNA samples are commonly preserved frozen until the intended study material is complete and can be analyzed simultaneously. Preservation time obviously depends on the study design, but short-term preservation for a few months seems reasonable for many current studies. However, a possible loss of DNA during the preservation period can bring a source of error to the results. In this study, we wanted to know, firstly, whether different preparations of saliva samples, i.e. whole saliva as such and, after centrifugation, the pellet and the supernatant, give different DNA yields, and secondly, whether the Preservation time affects the amount of DNA samples or whether oral bacteria selected.
section excerpts
Reference bacteria and culture conditions
Reference strains Filifactor alocis CCUG 47790, Porphyromonas gingivalis ATCC 33277 Parvimonas micra CCUG 46357, Prevotella intermedia ATCC 25611 and Fusobacterium nucleatum ssp. polymorphum NCTC 10562 were grown on brucella blood agar plates for 3 days. All strains were incubated under anaerobic conditions (10% H2, 5% CO2, 85% N2) at 37°C using the Anoxomat™ Mark II anaerobic gas filling system (Mart Microbiology, The Netherlands). Streptococcus mutans CCUG 11877 was also cultured in brucella blood
DNA concentration in purified samples
Table 2 shows the individual DNA concentrations of saliva samples from each study subject by DNA preservation time. DNA concentration did not significantly decrease during the 6-month preservation period in any of the samples (P>0.05), except for a small degradation of DNA purified from the saliva supernatant of some individuals.
As calculated for the study group, the mean (SE) concentrations of DNA (ng/μl) in total saliva were 152 (51.2) on day 0 and after 6 months 147.8 (50) in the sediment 135.3 (43) (day 0) and 133.6
Discussion
This prospective study demonstrated that the DNA yields of the three saliva preparations and the amounts of selected oral bacterial species did not significantly decrease during the 6-month preservation period at -80°C. All target species were found in all three saliva preparations, although values in total saliva and saliva sediment were consistently higher than those in the supernatant.
Our findings on lower DNA concentration and amounts of target species in saliva supernatant than in
Interest conflicts
The authors declare no conflicts of interest.
Acknowledgments
This study was funded by theKuwait University ResearchGrant SRUL01/14 and atKuwait Foundation for the Advancement of Scienceunder project code: 2011–5502-01.
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2019, Scientific Reports
Research Article
Subgingival microbiome in Chinese patients with generalized aggressive periodontitis compared to healthy controls
Archives of Oral Biology, Volume 101, 2019, pp. 92-99
The aim of the study was to profile the subgingival microbiome of Chinese adults with generalized aggressive periodontitis (GAgP) using the human oral microbe identification microarray (HOMIM) and compare the results with matched periodontal healthy controls.
15 individuals with GAgP and 15 age- and sex-matched periodontal healthy controls were included. Samples of subgingival plaques were collected from the deepest pockets of patients with GAgP and corresponding sites in controls, and then analyzed by 16S rRNA-based microarrays. Student's paired t-test was used to compare clinical parameters and the mean number of bacterial taxa detected between the two groups. Fisher's exact probability test and Wilcoxon Rank Sum were used to compare bacterial species among all samples. A multiple linear regression model was used for correlations between age, sex and bacteria with clinical parameters.
Of a total of 379 strains tested, 171 bacterial strains were detected in the subgingival plaques of GAgP patients, more than the 157 strains detected in the control group. The average number of subgingival bacterial taxa detected in the GAgP group was 68 (SD=21.06), while in the control group it was 45 (SD=21.60). 47 bacterial taxa were detected more frequently in the GAgP group, while 12 taxa were more prevalent in the control group. The significantly more prevalent and abundant bacterial taxa in the GAgP group included Filifactor alocis, Desulfobulbus sp., Fretibacterium sp., Porphyromonas gingivalis, Tannerella forsythia, Porphyromon as endodontalis, Peptostreptococcaceae spp., Parvimonas micra, Eubacterium nodatum, and Eubacterium saphenum. The most abundant taxa in the control group were Streptococcus spp. and Pseudomonas aeruginosa.
There are more bacterial taxa in the subgingival plaques of Chinese patients with GAgP than in healthy controls. F. alocis, Desulfobulbus sp., Fretibacterium sp., P. gingivalis and T. forsythia are strongly associated with GAgP. The elevated microbiological results may help dentists gain a better understanding of the subgingival GAgP microbiome.
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First case of renal abscess by Parvimonas micra
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Letter to the editor: “Siva cell-free DNA-based liquid biopsy as a potential biomarker for head and neck cancer”
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Evaluation of the performance of a new matrix-assisted desorption/ionization time-of-flight mass spectrometry, ASTA MicroIDSys system, in bacterial identification against clinical isolates of anaerobic bacteria
Anaerobic, Volume 61, 2020, Article 102131
Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was introduced for bacterial identification. The ASTA MicroIDSys system (ASTA, Suwon, Korea) is a new MALDI-TOF MS system developed for species identification of microorganisms. We evaluated the performance of MicroIDSys against clinical isolates of anaerobic bacteria.
A total of 370 unduplicated clinical isolates of anaerobic bacteria were tested in this study. Bacterial identification with MicroIDSys was performed with a direct smear method, and the measured spectra were analyzed using the respective software. MicroIDSys results were compared with Bruker Biotyper and 16S rRNA sequencing results.
The overall concordance rates for the 370 clinical isolates (34 genera and 99 species) were 95.4% (353/370) at the genus level and 91.6% (n=340) at the species level. Only 17 isolates were incorrectly identified at the genus level: five misidentifications and 12 misidentifications. The MicroIDSys system exhibited excellent performance in identifying clinically relevant bacterial species. Most Bacteroides isolates (98.0%, 99/101) and all Clostridium difficile (100%, n=11), Clostridium perfringens (100%, n=10), Finegoldia magna (100%, n=11 ), and Parvimonas micra (100%, n=10) were correctly identified at the species level.
The MicroIDSys system has proven to be useful in identifying anaerobic bacteria, especially clinically relevant species. This system may be useful in clinical microbiology laboratories as a primary tool for the identification of anaerobic bacteria.
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Dynamic behavior of cell-free mitochondrial DNA in human saliva
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Mitochondria contain their own genome that can be released into various biofluids such as blood and cerebrospinal fluid as cell-free mitochondrial DNA (cf-mtDNA). In clinical studies, blood cf-mtDNA predicts mortality and higher levels of cf-mtDNA are associated with physical and mental stress. However, the dynamics of cf-mtDNA has not been defined and it has not been examined whether it can be measured non-invasively like other neuroendocrine markers in saliva. Here, we report cf-mtDNA in human saliva and establish its natural dynamic behavior within the person over several weeks. In a small proof-of-principle cohort of healthy adults, we first developed an approach to rapidly quantify salivary cf-mtDNA without DNA isolation and demonstrated the existence of salivary cf-mtDNA. We then deployed this approach to perform an intensive repeated measures analysis of two healthy men studied at 4 daily time points for 53 to 60 consecutive days (n = 212 to 220 observations each) with parallel measurements of steroid hormones, mood self-reported diary, and health-related behaviors. Salivary cf-mtDNA exhibited a robust awakening response reaching up to two orders of magnitude 30-45min after awakening, varying from day to day and moderately correlated with the cortisol awakening response. In exploratory analyses, no consistent associations with daily self-reported mood/health-related behaviors were found, although this requires further examination in future studies. Dynamic variation in cf-mtDNA was inversely related to salivary interleukin 6 (IL-6), inconsistent with a pro-inflammatory effect of salivary cf-mtDNA. The highly dynamic behavior of salivary cf-mtDNA opens the door to non-invasive studies examining the relevance of mtDNA signaling to human health.
Research Article
DNA repair application for Streptococcus salivarius DNA-based identification of saliva samples exposed to ultraviolet light
Forensic Science International, Volume 306, 2020, Artigo 110077
Forensic samples are commonly influenced by several environmental factors, including ultraviolet (UV) irradiation; thus, forensic DNA repair applications (eg, PreCR™, Restorase®) were investigated, with a focus on short tandem repeat typing. However, current DNA-based tests are used for identification of human and bodily fluids. This study therefore aims to clarify the effectiveness of a DNA repair approach for DNA-based identification of Streptococcus salivarius from UV-damaged samples. Artificial UV-damaged genomic DNA of S. salivarius, saliva stains, and buccal swabs were used to evaluate the effects of DNA repair on the detection of S. salivarius DNA using the PreCR™ repair reagent. To evaluate forensic applications, we prepare simulated forensic samples by exposing them to environmental conditions. Melting curve analysis after real-time PCR was applied to qualitatively detect S. salivarius DNA with a specific melting peak of 80.5°C±0.4°C (n=10, mean ± 3SD). Single PCR was used for quantitative and qualitative analyses, while double PCR was used for qualitative detection of S. salivarius DNA. DNA repair experiments using UV-damaged artificial samples revealed a significant increase only in the quantitative value of genomic DNA samples by DNA repair. Furthermore, significant quantitative effects of DNA repair were not observed in all simulated forensic samples, indicating the limitations of DNA repair for DNA samples derived from real cells. Whereas differences in qualitative results (with or without detection) were generated for forensic sample simulations; thus, we considered the DNA repair strategy as an additional approach for S. salivarius DNA-based identification of environmentally damaged evidence.
© 2017 Published by Elsevier Ltd.